title: Short hairpin RNA gene silencing of prolyl hydroxylase-2 with a minicircle vector improves neovascularization of hindlimb ischemia.
abstract: In this study, we target the hypoxia inducible factor-1 alpha (HIF-1-alpha) pathway by short hairpin RNA interference therapy targeting prolyl hydroxylase-2 (shPHD2). We use the minicircle (MC) vector technology as an alternative for conventional nonviral plasmid (PL) vectors in order to improve neovascularization after unilateral hindlimb ischemia in a murine model. Gene expression and transfection efficiency of MC and PL, both in vitro and in vivo, were assessed using bioluminescence imaging (BLI) and firefly luciferase (Luc) reporter gene. C57Bl6 mice underwent unilateral electrocoagulation of the femoral artery and gastrocnemic muscle injection with MC-shPHD2, PL-shPHD2, or phosphate-buffered saline (PBS) as control. Blood flow recovery was monitored using laser Doppler perfusion imaging, and collaterals were visualized by immunohistochemistry and angiography. MC-Luc showed a 4.6-fold higher in vitro BLI signal compared with PL-Luc. BLI signals in vivo were 4.3×10(5)±3.3×10(5) (MC-Luc) versus 0.4×10(5)±0.3×10(5) (PL-Luc) at day 28 (p=0.016). Compared with PL-shPHD2 or PBS, MC-shPHD2 significantly improved blood flow recovery, up to 50% from day 3 until day 14 after ischemia induction. MC-shPHD2 significantly increased collateral density and capillary density, as monitored by alpha-smooth muscle actin expression and CD31(+) expression, respectively. Angiography data confirmed the histological findings. Significant downregulation of PHD2 mRNA levels by MC-shPHD2 was confirmed by quantitative polymerase chain reaction. Finally, Western blot analysis confirmed significantly higher levels of HIF-1-alpha protein by MC-shPHD2, compared with PL-shPHD2 and PBS. This study provides initial evidence of a new potential therapeutic approach for peripheral artery disease. The combination of HIF-1-alpha pathway targeting by shPHD2 with the robust nonviral MC plasmid improved postischemic neovascularization, making this approach a promising potential treatment option for critical limb ischemia.
authors.affiliation: 1 Department of Medicine and Radiology, Stanford University School of Medicine , Stanford, CA 94305.
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Lijkwan
authors.forename: Alwine A
authors.initials: AA
authors.lastname: Hellingman
authors.forename: Ernst J
authors.initials: EJ
authors.lastname: Bos
authors.forename: Koen E A
authors.initials: KE
authors.lastname: van der Bogt
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.forename: Nigel G
authors.initials: NG
authors.lastname: Kooreman
authors.forename: Margreet R
authors.initials: MR
authors.lastname: de Vries
authors.forename: Hendrika A B
authors.initials: HA
authors.lastname: Peters
authors.forename: Robert C
authors.initials: RC
authors.lastname: Robbins
authors.forename: Jaap F
authors.initials: JF
authors.lastname: Hamming
authors.forename: Paul H A
authors.initials: PH
authors.lastname: Quax
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D000792:Angiography; D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism; D006614:Hindlimb / Q000098:blood supply* / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000235:genetics* / Q000378:metabolism; D007511:Ischemia / Q000235:genetics* / Q000378:metabolism / Q000628:therapy; D051379:Mice; D018919:Neovascularization, Physiologic / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases